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2007; p2 - 11 



Review Article 



Stem cell therapy for neuropathic pain treatment 

Dario Siniscalco*, Francesco Rossi, Sabatino Maione 



'Department of Experimental Medicine - Section of Pharmacology "L. Donatelli", Second University of Naples 

* Dario Siniscalco, Department of Experimental Medicine - Section of Pharmacology "L. Donatelli", Second 
University of Naples. Via S. Maria di Costantinopoli, 16 - 80138 Naples, Italy. E.mail; dariosin@uab.edu 

Published online on 14 Nov 2007 



Abstract 

Pain initiated or caused by a primary lesion or dysfunction in the nervous system 
is defined as neuropathic pain. 

About 75 -150 million people in the United States are suffering for chronic pain 
disorder. Neuropathic pain has a great impact on the human wellbeing. It is very 
debilitating and often has an associated degree of depression that contributes to 
decreasing the quality of life. Moreover, the management of chronic pain is 
costly to the health care system. Pain is a national healthcare priority in US: the 
United States Congress has declared the present decade (2001-2010) as the 
"Decade of Pain Control and Research". 



Neuropathic pain is a very complex disease, involving several molecular 
pathways. Due to its individual character, its treatment is extremely difficult. 
Current available drugs are usually not acting on the several mechanisms 
underlying the generation and propagation of pain. 

Nowadays, pain research is focusing on newer molecular ways, such as stem cell 
therapy, gene therapy, and viral vectors for delivery of biologic anti-nociceptive 
molecules. These methods could provide a new therapeutic approach to 
neuropathic pain relief. 

Key words; neuropathic pain, stem cell therapy, gene therapy, virus vector 



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Pathophysiology of Neuropathic Pain 

Neuropathic pain is defined as pain initiated or 
caused by a primary lesion or dysfunction in 
the nervous system [1 ' 2 \ and several clinical 
symptoms are associated with it [3] . Most 
common are hyperalgesia (an increased 
response to a stimulus which is normally 
painful; patients with hyperalgesia perceive 
pain spontaneously) and allodynia (pain as a 
result of a stimulus which does not provoke 
pain; patients with allodynia do not feel 
constant pain, in fact in the absence of a 
stimulus there is no pain) [4] . Neuropathic pain 
can be triggered by central or peripheral nerve 
injury. Changes in the spinal cord or in the 
peripheral nerve, but also in the brain, have 
been reported, although these molecular 
alterations are still far to be clarified. 
Nociceptive signalling terminates in the spinal 
cord, the first centre involved in the 
controlling and processing of pain 
transmission. Indeed, in the dorsal horn of the 
spinal cord nociceptive afferent fibers 
terminate where the nociceptive neurons are 
located in the superficial lamina I (marginal 
layer) and in the lamina II (substantia 
gelatinosa). Interactions between nociceptive 
and non-nociceptive afferent pathways control 
the transmission of nociceptive information to 
higher centres in the brain [5] . 

Due to nociceptive input, such as peripheral 
nerve injury, the spinal cord anatomical 
structure is subjected to a re-organization. 
Indeed, the myelinated primary afferent fibers 
sprout into lamina II of the dorsal horn, 
establishing synaptic contacts with second- 
order neurons. In this way, they help to 
conduct the allodynic transmission [6] . 

Another change is a phenomena called "wind- 
up", a condition of central sensitization 
resulted from severe and persistent injury. In 
this condition, C-fibres are frequently sped on, 
releasing glutamate, and the response of the 
neurons of the dorsal horn spinal cord 
progressively increases [7 ' 81 . 



Glutamate is the major nociceptive excitatory 
neurotransmitter released from A-delta and C- 
fibres. Once released, glutamate is able to 
evoke fast synaptic potentials in dorsal horn 
neurons by activating the pre- and post- 
synaptic glutamate receptors. Among them, 
the ionotropic NMDA receptor is most 
involved in the events correlated with 
nociception [9], and with the maintenance of 
central sensitization and hyperexcitability of 
dorsal horn neurons. Activation of NMDA 
receptors increases the concentration of the 
calcium ion by the indirect activation of 
protein kinase C [10] . 

In the brain, the insular cortex is directly 
involved in the pain modulation. In this area, 
anti-nociceptive response is increased by the 
GAB A neurotransmission [11]. In particular, 
there is evidence that GABAa receptors 
modulate the nociceptive threshold affecting 
the noradrenergic bulbo-spinal projections 
from the insular cortex to the locus coeruleus, 
and GABAb receptors modulate the 
projections from cortex to amygdala 

Is the neuropathic pain a complete disease and 
not only the result of an other syndrome or 
injury? Interesting, newer molecular studies 
support this idea. Changes in DNA expression 
in the neuropathic pain syndrome have been 
observed. In response to peripheral noxious 
stimuli, dorsal horn neurons over-express the 
immediate early genes encoding transcription 
factors, such as c-jun and c-fos. These genes 
could be involved in cell death induction via a 
long-lasting cascade of transcriptional 
processes [12] . Indeed, the apoptotic genes 
mRNA expression levels of the bcl-2 cell 
death-associated family in the lumbar dorsal 
horn of the spinal cord of neuropathic rats are 
modified by peripheral nerve injury [13] . 

Following nerve injury, the afferent neurons 
(injured sensory neurons and their uninjured 
neighbours) close to the site of the injury 
increase their level of firing. This massive 
activity is called ectopic discharge, and it has 
also been proven in humans with neuropathic 
pain [14] . Altered expression of several types of 
sodium channels is responsible for the ectopic 



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firing after nerve injury, such as the voltage- 
gated sodium channels [15 ' 16] . The mechanisms 
responsible for the changes in the channel 
expression are not yet clear. Involvement of 
the neurotrophin (such as NGF, GDNF) 
supply has been suggested as a possibility . 



and mechanical allodynia in a pain model, the 
chronic constriction injury of the sciatic nerve, 
if administered locally on the site of nerve 



injury 



[17] 



The calcium channels may also contribute to 
the induction of hyperalgesia and allodynia 

[18] 



After peripheral nerve injury, sprouting of 
collateral fibres from sensory axons in the skin 
into denervated areas has been observed [19>20] . 
Neurotrophic factors and several cytokines, 
such as interleukin-1 (IL-1) and tumour 
necrosis factor-alpha (TNF-alpha), may be 
involved in the sprouting formation and in 
pathophysiology of neuropathic pain [21,22] . 

Classical pharmacological treatment 

Pain has a very complex nature. Nowadays 
there are not drugs for the neuropathic pain 
treatment acting in a complete and definitive 
way. 

o 

Currently, lidocaine, lamotrigine, 

acetaminophen, dextromethorphan, 
carbamazepine, gabapentin, valproic acid, 
opioid analgesics, tramadol hydrochloride, and 
tricyclic antidepressants are used for the 
classical pharmacological treatment of 
neuropathic pain. 

Clinical research is studying new direct-acting 
compounds to sodium and calcium channels 
since the ability of these channels to contribute 
to the development of neuronal 
hyperexcitability and the production of pain- 
associated behaviour. Lidocaine, a sodium 
channel blocker, is effective in the pain relief 
1231 , however, the available blockers are not 
specific between the several types of sodium 
channels. A private company is developing a 
new sodium channel blocker, Ralfinamide, for 
the potential treatment of neuropathic pain [24 ' 

25] 



More interesting, reduction of 



Specific antagonists for the neuronal calcium 
channel are able to reduce heat hyperalgesia 

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neuropathic pain associated with spinal cord 
injury in humans has been shown with 
intrathecal ziconotide, a marine-derived 
peptide [26] . 

Calcium flux is decreased by activation of the 
cannabinoid receptor subtype 1 . The synthetic 
cannabinoid CB1 receptor agonist Win 
55,212-2 decreases neuropathic pain 
behaviour, such as thermal hyperalgesia and 
mechanical allodynia [27] . 

As mentioned above, the main nociceptive 
neurotransmitter is glutamate. Inflammation 
and central sensitization are also controlled by 
NMD A glutamate receptors. NMD A receptor 
antagonists are able to attenuate neuropathic 
pain. Indeed, the NMDA receptor antagonist 
MK-801 has a potent anti-nociceptive effect 
[28, 29, 30]^ but du£ tQ - ts jjjgjj tox j c p r0 p er ties 

and low safety margins it is not available for 
clinical use on human patients. Nevertheless, 
amantadine, dextromethorphan, ketamine, and 
memantine are commercially available 
NMDA-receptor antagonists. The opioids 
methadone, dextropropoxyphene and 
ketobemidone are also NMDA-antagonists, as 
well as the triciclic antidepressant amitriptiline 
[3i, 32] NMDA-receptor antagonists in 
combination with opioids might represent a 
new class of analgesic and might have 
potential as a co-analgesic; NMDA-receptor 
antagonists help to enhance development of 
tolerance to opioid analgesics [33] . 

In pain transmission, glutamate activates also 
group I metabotropic receptors (mGluRs). 
Peripheral and central mGluR5 receptors are 
responsible of the nociceptive transmission 
observed during post-operative pain [34] . 
MPEP, the potent and selective antagonist for 
metabotropic glutamate receptor subtype 5 
(mGlu5), is able to prevent the development of 
thermal hyperalgesia, transiently reduce 
mechanical hyperalgesia in neuropathic rats, 
and prevent the over-expression of pro- 
apoptotic genes in dorsal horn spinal cord 



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neurons . This subtype of metabotropic 
glutamate receptors could represent the 
prototype of new potential drugs in pain 
treatment; however, due to the complex role of 
glutamate in the nervous central system, 
blockade of glutamate receptors is associated 
with several side effects. 

The typical mu-opioid analgesics, such as 
morphine, can be relatively ineffective in 
treating neuropathic pain since different 
opioids can produce analgesia by affecting 
different pain pathways [35] . 



responsiveness of dorsal horn sensory neurons 
and to hyperalgesia and allodynia [16 ' 41 ' 42 ' 43 ' u ' 
45 ' 461 . Gene silencing by the use of antisense 
oligonucleotides, a novel molecular 
pharmacological approach, causes a decrease 
in pain-related behaviour. 

Nicotinic receptors, P2X receptors, 5-HT1A 
receptors, NMDA glutamate receptors and 
opioid receptors have been successfully used 
as target for antisense knock-downing 
strategy, showing a decrease in nociceptive 
behaviour [47 ' 48 ' 49 ' 50 ' 51 ' 52] . 



Likely, the optimal classical drugs in the 
treatment of neuropathic pain are the 
anticonvulsant gabapentin, and its successor 
pregabalin [36 ' 37 ' 38 ' 39] . They are able to 
decrease the hyperexcitability of dorsal horn 
neurons induced by tissue injury, but their 
mechanism of action is still unclear. 
Interesting, they have only an effect in a 
condition of sensitization of a nociceptive 
pathway. 



Molecular methods for neuropatl 
treatment 




Newer molecular methods, such as gene 
therapy and viral vector for the delivery of 
biologic anti-nociceptive molecules, could 
represent a novel therapeutic approach to the 
neuropathic pain treatment [40] . 

Following peripheral nerve injury, spinal re- 
organization and changes in the excitatory or 
inhibitory pathways controlling neuropathic 
pain development are correlated with altered 
gene expression. Novel molecular 
pharmacological strategy is directed toward 
the control of the gene up- or down-regulation. 
Antisense knock-down strategy could 
represent a novel approach to the neuropathic 
pain therapy in the nearest future. As next 
step, antisense research has to elucidate the 
pharmacodynamics, pharmacokinetics and 
distributions of antisense oligonucleotides. 

Among the genes showing altered expression 
in neuropathic pain, several sodium and 
calcium channels contribute to the hyper- 

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Immediate early genes, such as c-fos, are over- 
expressed in dorsal horn neurons of the spinal 
cord after peripheral nerve injury. Also in this 
case, intrathecal administration into the 
lumbar region L1-L5 of c-fos antisense 
oligonucleotides has shown a role played by 
the c-fos gene in neuropathic pain [53] . 

Viral vector technology to delivery anti- 
nociceptive molecules could represent a novel 
therapeutic strategy. Dorsal root ganglion 
neurons transduced with replication- 
incompetent herpes simplex virus (HSV-) 
based vector, encoding the GAD67 isoform of 
human glutamic acid decarboxylase, are able 
to produce GAD and release GABA, reducing 
neuropathic pain following a spinal cord 
injury [54] . Constitutive GABA expression via 
recombinant adeno-associated virus producing 
GAD65 attenuates neuropathic pain [55] . It has 
been demonstrated that virus encoding human 
pre-proenkephalin (hPPE) are able to decrease 
the activation-levels of nociceptors by 
capsaicin treatment in mice and macaques [56] . 

Coupling antisense knock-down and viral 
vector technology is showing promising 
results. Virus delivering antisense cDNA 
versus calcitonin gene-related peptide 
precursor (ACGRP) decreases C-fiber 
hyperalgesia due to the application of 
capsaicin on the skin in mice [561 . 

Potentially, all the molecules, such as 
neurotrophines, having nociceptive effects 
could be delivered by adenovirus. Candidate 
gene products include directly analgesic 

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molecules, as well as molecules that are able 
to interfere with pain-associated biochemical 
changes in pain pathways. Recombinant 
adenovirus encoding NT-3, BDNF, GDNF, or 



Semaphorin3A into animal models of 
neuropathy showed good results for 

■ • ,• r [57, 58, 59, 60, 61, 62] 

neuropathic pain relief L J . 
Intrathecal delivery of the adenovirus- 
mediated IL-2 gene has a relatively long anti- 
nociceptive effect [63] . 

Non-invasive gene delivery systems could be 
usefully used for targeting peripheral nervous 
system pathologies. Subcutaneous peripheral 
injection of plasmid DNA complexed with a 
non-viral cationized gelatin (CG) vector led to 
transgene expression in rat lumbar dorsal root 
ganglia [64] . 

Stem cell therapy 

Nowadays, stem cell therapy represents the 
great promise for the future of molecular 
medicine. Several diseases can be slowed or 
even blocked by stem cell transplantation. 
Stem cells could be neuroprotective in a 
variety of nervous system injury models. As 
neurodegenerative disease, also neuropathic 
pain undergoes to stem cell therapy [40] , even if 
the state of the art is still poor of basic and 
clinical research. 

Marrow mononuclear cells containing mixed 
stem cell populations have been intravenous 
used in neuropathic rats showing recovery 
from pain [65] . 

Stem cell implantation could be a possible 
solution for spinal cord injury. Stem cells have 
the ability to incorporate into spinal cord, 
differentiate, and to improve locomotor 
recovery 



[66] 



Despite ethical problems, it has been 
demonstrated that human embryonic neural 
stem cells can promote functional 
corticospinal axons regeneration and synapse 
reformation in the injured spinal cord of rats. 
The action is mainly through the nutritional 
effect of the stem cells on the spinal cord. 

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Transplanted cells were found to migrate into 
the lesion, but not scatter along the route of 
axon grows. The cells differentiated into 
astrocytes or oligodendrocytes, but not into the 
neurons after transplantation [67] . 

Spinal progenitor cells intrathecally 
transplanted in neuropathic rats are able to 
alleviate neuropathic pain [ 68 ]. Murine neural 
stem cells (NSCs) homografted onto the 
injured spinal cord improved motor behaviour 

[69] 

How do stem cells work? Stem cells 
transplanted following spinal cord injury are 
able to reduce allodynia and improve 
functional recovery if they produce more 
oligodendrocytes than astrocytes [70] . 
Serotonergic neural precursor cell grafts are 
able to reduce hyperexcitability caused by 
spinal injury [71] . Neuropathic pain causes a 
decrease in the number and activity of 
GABAergic neurons, the spinal progenitor 
cells show glutamic acid decarboxylase 
immunocompetence, in this way they can 
supply the decreased GAB A profile [70 ' 721 . 

Is the stem cell differentiation the key for the 
pain care? Or do they provide several 
molecules with analgesic action? Indeed, 
using of genetically engineered stem cells 
expressing anti-nociceptive molecules or 
trophic factors seems to be an useful tool in 
neuropathic pain relief. Stem cells could be 
used as biologic "minipumps" to chronically 
deliver anti-nociceptive molecules close to the 
pain processing centers or the sites of injury 

[73, 74] 

Besides genetic engineering, stem cells 
applied to the site of the injury could provide 
trophic factors directly in situ, by this way 
acting as anti-nociceptive drug. 

Among the stem cell population, 
mesenchymal stem cells (MSCs) rise probably 
best potential good results in pain-care 
research. These cells are a population of 
progenitor cells of mesodermal origin found in 
the bone marrow of adults, giving rise to 
skeletal muscle cells, blood, fat, vascular and 

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urogenital systems, and to connective tissues 
throughout the body [75,76] . MSCs show a high 
expansion potential, genetic stability, stable 
phenotype, can be easily collected and shipped 
from the laboratory to the bedside and are 
compatible with different delivery methods 
and formulations [77] . In addition, MSCs have 
two other extraordinary characteristics: they 
are able to migrate to sites of tissue injury and 
have strong immunosuppressive properties 
that can be exploited for successful autologous 
as well as heterologous transplantations I78] . 
Besides, MSCs are capable of differentiating 
into neurons and astrocytes in vitro and in 
vivo [79] . Recently, MSC injection has shown 
good results for amyotrophic lateral sclerosis 
treatment in human [80] . They are able to 
improve neurological deficits and to promote 
neuronal networks with functional synaptic 
transmission when transplanted into animal 
models of neurological disorders [81] . 

MSCs have been observed to migrate to the 
injured tissues and mediate functional 
recovery following brain, spinal cord and 
peripheral nerve lesions, suggesting that 
MSCs could modulate pain generation after 
sciatic nerve constriction [821 , although the 
underlying mechanisms by which MSCs exert 
their actions on pain behavior is still to be 
clarified. 

We are currently studying the use of human 
mesenchymal stem cells (hMSCs) for 
neuropathic pain treatment in rodents. hMSCs 
micro-injected into specific nuclei involved in 
pain processing were able to completely 
abolish pain-like behaviour in neuropathic 
mice (Siniscalco, 2007, unpublished data). 

Recently, Dr Stephen Richardson of the 
University of Manchester has developed, 
under patent, a cell-based tissue engineering 
approach to regenerate the intervertebral disc 
at the affected level in the low back pain 
(www.ls.manchester.ac.uk/ukctr). This is 
achieved through the combination of the 
patients' own mesenchymal stem cells and a 
naturally occurring collagen gel that can be 
implanted through a minimally-invasive 
surgical technique. Hopefully, once implanted 

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the differentiated MSCs would produce a new 
tissue with the same properties as the original 
and would both treat the underlying cause of 
the disease and remove the painful symptoms. 

Conclusions 

Neuropathic pain has a great impact on the 
quality of life, reducing human wellbeing. 
Management of chronic pain is very costly to 
the health care system. Since 75-150 million 
people in the United States have a chronic 
pain disorder [40] . The United States Congress 
has declared the present decade (2001-2010) 
as the "Decade of Pain Control and Research", 
making pain a national healthcare priority. 

Neuropathic pain involves several molecular 
pathways and is a very complex disease. It has 
an individual character, making its treatment 
extremely difficult. Currently, available 
treatments address the pain-symptoms using a 
combination of painkillers. None of these is 
ideal as they only treat the symptoms and 
temporal pain properties, not the cause, and 
limited long-term success. 

Novel molecular methods, such as antisense 
strategy, gene therapy, and virus therapy, are 
acting on the several mechanisms underlying 
the generation and propagation of pain. More 
recently, preliminary clinical evidence 
suggests that stem cell therapy could provide 
best results, this strategy could be the 
definitive pain-relief drug for the next future. 

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